CCK and other peptides modulate hypothalamic norepinephrine release in the rat: Dependence on hunger or satiety

0361~9230/86 $3.00 + .OO

Brain Research Bulletin, Vol. 17, pp. 583-597, 1986.0 Aakho International Inc. Printed in the U.S.A.

CCK and Other Hypothalamic Release Dependence on

Peptides Modulate Norepinephrine in the Rat: Hunger or Satiety

R. D. MYERS,’ IX S. SWARTZWELDER,2 J. M. PEINADO, T. F. LEE,3 J. R. HEPLER,* D. M. DENBOW AND J. M. R. FERRER6 Departments of Psychiatry and Pharmacology, and Center for Alcohol Studies University of North Carolina, School of Medicine, Chapel Hill, NC 27514

MYERS, R. D., H. S. SWARTZWELDER, J. M. PEINADO, T. F. LEE, J. R. HEPLER, D. M. DENBOW AND J. M. R. FERRER. CCK and other peptides modulate hypothalamic norepinephrine release in the rat: Dependence on hunger or satiety. BRAIN RES BULL 17(4) 583-597, 1986.-The purpose of this investigation was to determine the functional relationship between putative satiety peptides and endogenous norepinephrine (NE) activity in the hypothalamus. Permanent guide cannuiae for push-pull perfusion were implanted stere.otaxicalIy in Spm~e-~wIey rats so as to rest above the medial or lateral hypoth~amus (LH). Post-operatively, the animals were either satiated with food and water, both available ad fib, or fasted for lg-22 hr prior to an experiment. To perfuse a site in the LH, paraventricular (PVN) or ventromedii nucleus (VMN), a concentric 29-23 ga push-pull cannula system was lowered to a pre-determined site, in most cases after catecholamine stores had been pre-labeled with [3H]-NE. During control tests, an artificial CSF was perfused at a rate of 20-25 pl/min for 5-8 min with a 5 min interval between each sample. The addition of cholecystokinin (CCK) in a concentration of 2.0-6.0 n&l to the CSF perfused in PVN or VMN of the satiated rat enhanced the efflux of NE; however, in the fasted animal CCK often suppressed the catecholamine’s release. Perfused in the LH, CCK exerted opposite effects, typicalfy augmenting NE output when the rat was fasted but not affecting the amine’s activity during the sated condition. Proglumide (1.2 fig/PI) attenuated CCK’s effect in releasing NE when the antagonist was perfused in the PVN of the satiated rat. Similar experiments in which neurotensin (NT) was perfused in the LH, PVN and VMN revealed virtually the same inverse effects on NE release in the fasted and satiated rat, which again were anatomically specific. Finally, insulin and 2-deoxy-D-glucose (2-DG) exerted similar state-dependent effects on the release of NE within LH and PVN. Overall, the results suggest that CCK or other neuroactive peptide could serve as a “neuromodulator” of the pre-synaptic release of NE within classical hypothalamic structures which are thought to underlie both hunger and satiety. The state-dependent nature of the peptides’ activity on the noradrenergic feeding mechanism implies that these substances constitute a pivitol portion of the profile of factors which impinge function~iy upon the hypothetic neurons responsible for the feeding response and its cessation. Neurotensin Cholecystokinin (CCK) Norepinephrine (NE) Paraventricular nucleus (PVN) Lateral hypothalamus (LH) Satiated state Neuromoduiators Peptides Brain

IT has been over 20 years since the first reports began to appear which described the phenomenon of spontaneous feeding produced by a catecholamine applied directly to the hypothalamus of the satiated rat [21]. Since that time, literally hundreds of scientific publications have documented the likely role of noradrenergic pathways in the brainstem underlying the feeding mechanism

(NT) Insulin 2-deoxy-D-glucose (2-DG) Ventromedial nucleus (VMN) Fasted state

A3BREVIATIONS pCPA DHT ICV

parachlorophenylalanine dihydroxytryptamine intracerebroventricular

[40,57].

‘Requests for reprints should be addressed to R. D. Myers, Bowles Biomedical Research Laboratory, Medical Research Building A, 218H, University of North Carolina School of Medicine, Chapel Hill, NC 27514. Tresent address: Division of Neurology, Duke University Medical Center, VA Hospital, Durham, NC. ‘Present address: Department of Zoology, University of Alberta, Edmonton, Canada. ‘Present address: Neurobioiogy Program, University of North Carolina School of Medicine, Chapel Hill, NC. “Virginia Polytechnic Institute and University, Blacksburg, VA. ‘Department of Physiology, University of Oxford, Oxford, U.K.

583

584

An overview of representative early findings through 1975 1581 which support the role of catecholamines in the central control of feeding is as follows: First, catecholamines are present endogenously in lateral, media1 and other areas of the hypothalamus implicated in the control of feeding. Second, the local application of catecholamine agonists, including norepineph~ne (NE) and epinephtine (E) induces spontaneous feeding in the rat, monkey and many other species. Third, catecholamine-induced feeding can be selectively attenuated by adrenergic blocking agents which attach to the alpha-adrenergic receptor. Fourth, a lesion to the brainstem pathway which depletes forebrain catecholamines interferes with the regulation of food intake. Fifth, a lesion produced by the catecholamine neurotoxin, 6hydroxydopamine (6OHDA) produces a set of multiple impairments to ingestive behavior when the neurotoxin is infused into catecholaminergic pathways, the hypothalamus and cerebra1 ventricle. Sixth, food deprivation influences not only the content of NE in the hypothalamus of the rat but also the synthesis or turnover of dopamine (DA) and NE. Seventh, NE and DA are released from certain structures of the hypothalamus of the rat and monkey during ingestive behavior. Overall then, the evidence is impressive that NE could serve as a transmitter in the hunger-satiety mechanism within the hypothalamus. In fact, NE fulfills the essential criteria required for an endogenous factor to be considered as a neurotransmitter in this structure 1571. In the last decade, many new experiments have provided additional support for this viewpoint. As reviewed by Leibowitz [40], numerous advances have occurred along many research fronts, including the localization of the area of maximal sensitivity to NE to the paraventricular nucleus (PVN), differentiation of NE binding in hy~th~~ic tissue as well as the characteristics of a physiologically induced release of NE from the hypothalamus of the conscious, freely moving animal [40, 44, 481. Substances other than catecholamines, which are also present endogenously in hypothalamic tissue, likewise have been implicated historically in the feeding mechanism [7 1,791. For example, serotonin (S-HI) has been considered as a potential satiety factor in the periphery as well as in CNS (e.g., [5]). A large amount of indirect pharmacological evidence on the role of 5-HT has centered on studies involving the systemic administration of pCPA or tryptophan and on 5,6-DHT or 5,7-DHT given by one of several central routes (e.g., 1601). However, the latter serotonergic neurotoxins and 6OHDA given ICV either impair food intake to some degree. or fail to alter it, depending on the strain of rat tested (e.g., [60]). Nonetheless, on the basis of microinjection studies, it is believed that 5-HT receptors in the PVN could in some way counteract noradrenergic receptors to inhibit feeding [27,54]. This implies that endogenous 5-HT would be released from serotonergic terminals to stimulate “satiety neurons,” a possibility not yet demonstrated. Opioid peptides comprise another class of compounds that also have been implicated indirectly in the central mechanism for feeding. Pharmacological results based on a systemically administered opiate antagonist, naloxone, show that the antagonism of opiate receptors attenuates the “feeding drive” 1531. On the other hand, several opiate agonists and mixed ~onist/an~gonist drugs evoke eating when injected peripherally ]53]. Moreover, the intrahypothalamic infusion of certain opiate agonists can produce feeding (e.g., [g7]). Other substances postulated over the years to play one or

MYERS ET Al,. another role in the central control of feeding include GABA and other amino acids, free fatty acids, acetylcholine, prostaglandins, bombesin, calcium ions, calmodulin, calcitonin, cholecystokinin and the most powerfully acting substance discovered yet, neuropeptide Y [26, 35, 52, 821. However, unlike the vast store of observations accumulated on NE, other putative hunger and satiety factors have not been explored so fully in terms of specific experiments such as: agonist-antagonist actions; neurotoxic and other (CNS lesions; generalization across species from bird to primate; and finally, the stimulus-induced release of the putative factor from the hypothalamus of the conscious animal during feeding, nutrient deficit and other physiologically relevant conditions. The latter is perhaps the most impo~~t functional consideration from the perspective of transmitter criteria [57]. During the last decade, the experimental focus in our laboratory has shifted somewhat away from an exclusive emphasis on the pharmacological manipulation of hypothalamic nuclei and other structures [35,37] toward the characterization and qu~ti~cation of endogenous NE and other factors during a physiological event associated with hunger or satiety. The reasons for this focus are two-fold: First, it is becoming increasingly more difftcult to specify the precise action of a compound on cells of the hypothalamus in modifying an ingestive response. Second, it is essential to know what transpires cellularly when a factor enters into a tissue compa~ment of the hypothalamus or impinges upon the catecholamine systems involved in feeding. The specific purpose of these studies, therefore, has been to elucidate the functional interaction of selected, potent peptides and other substances with noradrenergic neurons within the hypothalamus [61]. In these experiments, the local effect of cholecystokinin (CCK), neurotensin (NT), insulin and 2-deoxy-D-glucose (2-DG) has been examined on NE activity in anatomical regions of the hypothalamus that mediate both feeding and satiety. GENERAL

METHOD

Throughout the last decade, the methods used in our laboratory have followed the same general principles 156,591. Essentially, a specific site in the hy~thal~us of the unrestrained animal is perfused repeatedly with an osmotically balanced, artificial CSF, with individual samples collected in serial fashion. At a given point in the series of perfusions, a substance is added to the perfusate in order to determine its action on the kinetics of release of a neurotransmitter such as NE, DA or 5-HT 142,441. Adult Sprague Dawley rats of either sex are housed individually in a colony room at an ambient temperature of 2123°C. The room is maintained on a 12 hr reversed light cycle with white light on from 2200-1000 hr and the remaining 12 hours illuminated by red fluorescent lighting. Standard laboratory chow and water are provided ad lib to the animals except during an experimental session. In this case, either a period of up to 18 to 22 hours of food deprivation is imposed, or the animal is simply permitted to feed without restriction. An interval of no longer than 24-48 hours is usually permitted to elaspse between each experiment since a longer period may result in technical difftculties with the push-pull perfusion [59].

Under anesthesia, stainless steel guide tubes are surgically implanted either unilaterally or bilaterally according to

CCK AND NOREPINEPHRINE

RELEASE

standard stereotaxic procedures [55] with the incisor bar set at 5.0 mm above the zero plane. Each guide tube is always positioned above the intended sites of perfusion, in order to minimize damage to the tissue. The tube is then fitted with an indwelling stylet in order to prevent its occlusion. Generally, the coronal coordinates [73] for studies of hypothalamic function range between AP 4.0-7.5 with the lateral coordinates being 0.3-2.5 from mid-line. After the cannulae are affixed to the skull with cranioplastic cement, a polyethylene cap is screwed onto the pedestal in order to protect the cannula and to maintain a sterile preparation. Push-Pull

Perfusion

After 7 to 10 days elapses post-operatively, the rat is placed in an individual Nalgene test chamber. In most of the experiments, the perfusion site is labeled with [3H]-NE microinjected in a volume of 0.5-1.0 ~1 over an interval of 30 sec. In many experiments, either Y-DA or 14C-5HT has been included in the NE label; however, only 13Hl-NE results will be described here. After a period of 15-30 min has elapsed following the tracer labeling, a concentric push-pull cannula assembly is lowered to the site to be perfused. Ordinarily, the first depth of perfusion is 1.0 mm below the tip of the guide tube. All of the experimental test perfusates as well as control samples are collected at this first site before the next depth is perfused, usually in 0.5-1.0 mm increments [62]. The pet&sate consists of an artificial CSF containing Na+ 127.7 mM; K+ 2.6 mM; Ca++ 1.3 mM; Mg++ 0.9 mM; and Cll 132.5 mM [55]. To retard the degradation of the amine, 0.01 mg/ml ascorbic acid is typically added to the CSF in most of the experiments, after which the solution is passed through an 0.22 pm Swinnex millipore filter into a pyrogen free vial [42]. Each perfusion is carried out at a rate of flow of 20-25 pl/min and lasts for 5-8 min with 5 min usually intervening between successive perfusions. After the third or fourth perfusion, when the level of radioactivity in the series of perfusates has begun to stabilize, a peptide or other test compound is incorporated into the CSF which is then perfused at the same site. In selected cases, excess K+ ions are added to the CSF perfusate in a concentration of 25 mM in order to validate the integrity of the site in terms of an enhanced release of catecholamine which is produced by this ion species [62]. An experiment is always terminated immediately if the sample of perfusate is discolored, contains bubbles, or if the flow rate changes. In the control experiments, samples of perfusate are collected successively without the addition of any compound to the perfusate in the mid-point of the test run. Assay

Procedures

Two procedures for assaying push-pull perfusates collected at discrete anatomical loci are currently used in our laboratory: (1) radio-tracer, and (2) high performance liquid chromatography with electrochemical detection (HPLCEC). Radio-tracer technology for analyses of the effect of a physiological or behavioral stimulus on the activity of a catecholamine or other neurotransmitter constitutes an extremely powerful one. It is also a valid experimental tool, particularly when used in conjunction with thin layer layer chromatographic (TLC) separation of transmitter metabolites [47]. One criticism of the tracer technique for push-pull perfusion studies revolves about the possibility that the nu-

585 elide labels monoamine stores other than neuronal stores [74]. According to this view, when a physiological or behavioral event stimulates the release of an amine into the extracellular space where it is collected in the perfusate “washing the site,” the origin of radioactivity could be non-neuronal. Presumably, a primary source of extra-neuronal release would be vascular, e.g., an arteriole. For several reasons, this criticism does not appear to be applicable in this instance. First, other assay systems that could be used as an alternative, such as HPCL-EC or electron capture-GC, would similarly detect the same amine as it is released regardless of origin, neuronal or extra-neuronal. Second, it is unlikely that an amine-related change in blood vessel walls, for example, would predominate over a series of synaptic events in neurons of the medial or lateral hypothalamus in determining a feeding response or its termination. Third, in double-labeled experiments in which W-DA or [3H]-NE are microinjected together at the same site, a partial or complete dissociation of release of the radiolabeled amines can occur at that perfusion site which depends solely upon a specific functional stimulus [41,78]; this thereby excludes a non-specific efllux of the label. Fourth, an experiment employing push-pull perfusion with a precursor such as [3H]-tyrosine has the singlular disadvantage of requiring an inordinately large amount of labeled precursor. In fact, in experiments undertaken in our laboratory in the early 1970’s, the recovery of NE from labeled tyrosine was not only very low but sometimes undetectable, an outcome not unexpected since NE is three enzymatic steps removed from this precursor [46]. In the present experiments utilizing the labeled catecholamine, each sample of perfusate collected during a 5 min interval was transferred to a mini-vial containing 3.0-5.0 ml of PCS fluor (Amersham). The [3H]-NE activity of each individual sample was counted in a Tracer Analytic Mark III Scintillation Spectrometer and corrected to dpm automatically by microprocessor. In selected samples, a small aliquot of perfusate also was analyzed by two-dimensional TLC [47], to verify both the presence of metabolites of the catecholamine and the decline in activity of NE in succeeding samples of perfusate due to its metabolism [65]. In the experiments in which an HPLC-EC assay system was used, sodium metabisullite was added as an anti-oxidant to the CSF perfusate in a concentration of 0.01 pg/ml. After the perfusate was collected in a microcentrifuge tube placed on dry ice, each sample was lyophilized immediately in a freeze dryer and stored at -20°C until analysis. Prior to injection onto the HPLC column, the samples were reconstituted in 10 ~1 of 0.05 M HClO, containing an internal standard, isoproteronol, in a concentration of 0.8 picograms 10 ~1. For the present experiments, the HPLC system consisted of a Constametric model III pump, a Rheodyne loop injector and an LC-4 amperometric detector equipped with a ‘IL-8A glassy carbon working electrode. The oxidation potential was +0.85 V with a sensitivity of 0.50 nanoamps per volt. The output was recorded simultaneously on a Hewlett Packard 3390A integrator and an Omniscribe stript chart recorder. The HPLC column used was a 5.0 pm Biophase GDS with a 5 pm Biophase ODS Guard column. The mobile phase contained 0.12 M citric acid, 350 mg/l NaOH, 150 mg/l NaFDTA and 110 n&l Na-Octyl-SO,, and was degassed and filtered through a 0.22 pm vacuum filtering apparatus (Millipore). The final pH of the mobile phase was adjusted to 3.15 with concentrated NaOH after 80 ml HPLC grade acetonitrile and 1.8 ml triethylamine had been added.

586

MYERS ET-AL.

CCK 4 NE RELEASE FASTED \

[SATIATED

TABLE

1

SIGNIFICANT INCREASE (f), DECREASE (J) OR NO CHANGE (0) IN NE RELEASE FROM SITE IN DIENCEF’HALONPERFUSED WITH CCK. INDEPENDENT OF DOSE, WHEN RAT WAS SATIATED FOOD-DEPRIVED FOR 22 HOURS

OR

NE Response to CCK Rat (depth)*

site

AP Plane

Satiated

Fasted

Lateral Hypothalamus A(t) B(l)

B(2) C(1) A(2) D(2) E(1)

F(1) F(2)

DLH DLH DLH DLH LH LH LH LH LH

5.0 4.0 4.0 6.0 5.0 4.5 5.0 5.0 5.0

0 0 0

T

! i I, 0 f

0

0 -_ -

VMN-PVN VMN

5.0

VMN

5.0

wt

VMN

5.5

J(3) l(3) Q(I) H(1) R(1) K(1) J(I)

VMN VMN PVN PVN PVN

5.5 5.5 6.5 5.5 6.0

PVN PVN

6.5 6.0

G(2) H(2)

0

T 1

0 -

-7 I‘ i T

i -

-

1

0

0

1

i f *

DMN and Other Sites C(3)

FIG. I. Anatomical “mapping” of hy~thaiamic sites at which CCK was perfused in a concentration of 0.05 &min in rats which were 18-24 hr food-deprived (left) or fully satiated (right). Release of [‘H]-NE from each site is denoted as increased (A), decreased (v) or unchanged CO) during CCK perfusion.

6.0 5.5 5.0 5.5

0 i

i

B(3)

DMN DMN DMN/VMN DMN~VMN MM

4.0

?‘

0

M(2) N(1) N(2)

MM LHIPVN LH/DMN

X.5 55 .T.5

0

I

CF OT AVN MFB PAG ARCIVMN

1

I

5 .5 6.0 5.5

0

J(2) G(1) L(1)

N(3)

Data Anulysis

C(2)

At the conclusion of the series of experiments, the anatomical position of each guide cannula and the sites of posh-pull .perfusion were verified in the rat according to standard histological procedures [57]. After 0.9 percent saline followed by a buffered 10 percent formalin solution was perfused either through the thoracic aorta or transcardially, the brain was removed from the skull. Histological sections were cut on a cryotome in the coronal plane at 7.5 microns, then mounted and stained according to a modified Kltiver-Barrera or other procedure [42, 44, 641. Each site of perfusion was determined by light microscopy with a multiobjective microprojector. Subsequently, composite ” histological maps” of each locus of perfusion were constructed in the coronal plane for detailed anatomical analysis 144, 48, 571. Observations on the eMux of radiolabeled catecholamine from the site of hypothalamic perfusion were analyzed by the proportional method of Hail and Turner 1231.This procedure is used because the absolute values of dpm in each sample of perfusate often varies between each experiment [41]. Given that the dpm value of the sample collected immediately be-

O( I ,2) D(I) M(I) Pm

4.3

3.0 5.0

0

T * 0 -

0 f -

0 0 i

Abbreviations: AVN. antero-ventral thalamic nucleus: ARC, arcuate nucleus; CF. columns of fornix: DLH. dorsolateral hypothalamus; DMN. dorsomedial nucleus: LH, lateral hypothalam~s~ MFB, median forebrain bundle; MM, mamillary bodies; OT. optic tract; PAG, periaqueductal grey: PH. posterior hypothalamus; PVN, paraventricular nucleus; VMN. ventromedial nucleus. The designation of DMN/VMN, LH/PVN. LH/DMN and ARCiVMN indicates that the tip of the perfusion cannulae encompassed a portion of both structures. *“Depth” denotes level of perfusion site in same animal.

fore the test substance serves as the proportional baseline value of 1.00, a change in the magnitude of [“HI-NE efflux into the push-pull perfusate was categorized according to previousiy adopted stringent criteria [44] as follows: (1) a

CCK AND NOREPINEPHRINE

587

RELEASE

SATIATED

FASTED

PARAVENTRICULAR

N.

2.0g 1.5. I? iii Qz 1.0. s $0.5.

.

-40

0

40

.

.

80 -40 0 TIME (MINUTES)

.

.

40

.

.

80

FIG. 2. Proportional [3H]-NE release from perfusion site in AP 6.5 within PVN of a representative rat when fully satiated (left) or fasted for 22 hr (right). CCK (0.05 &min) was perfused (histological inset) at zero time and excess K+ ions (25 mM) at 50 min time (right). Asterisk (*) denotes statistically significant change (pSO.01).

change in efflux of amine was considered statistically significant if it differed from the control value by +2.31 S.E. which has a probability of co.01 [44]; (2) amine release was deemed immediate if the proportional dpm value was greater than 1.00 in the sample collected during the perfusion of the test substance; (3) the release of amine was considered “delayed” if the proportional dpm value of the sample of perfusate obtained following the test perfusion was greater than that of the sample containing the test substance; and (4) no change in r3H]-NE release was designated if the slope of the activity failed to exceed the S.E. of the control values. CCK “MODULATES”

NOREPINEPHRINE

ACTIVITY

With the advent of the announcement of Gibbs et al. in 1973 [ 171on the satiating effect of CCK given systemically, a number of researchers have provided impressive support for the idea that endogenous CCK can function as a satiety hormone [3]. This viewpoint has been substantiated by pharmacological experiments in which CCK has been administered to the chicken, rat, sheep, pig, monkey, mouse and man 111, 51, 801. Based on experiments on the totally or partially vagotomized rat, it has been proposed that the inhibition of gastric emptying into the duodenum is possibly a critical event in the action of CCK in producing satiety [80]. Alternatively, CCK circulating in the bloodstream also could act peripherally if the peptide were taken up by receptors in the atferent portion of the vagus nerve. Another possibility is that CCK operates directly within the CNS, primarily on hypothalamic neurons comprising a portion of the feeding system 111, 42, 511. A series of papers published by Della-Fera and colleagues [8-lo] have provided compelling evidence that the CSF spaces in the brain, at least in part, subserve the satiating effect of CCK. A picomolar quantity of CCK infused into the ventricular lumen of the sheep inhibits feeding; conversely, CCK antibody, which serves to reduce the peptide’s activ-

ity, augments the intake of food in the sheep when it is infused similarly [lo]. Nevertheless, the satiating property of CCK has not been without controversy [3]. For example, when given by the ICV route, CCK can produce a motor impairment [45] or disturbance to respiratory and cardiovascular function [72]. However, centrally administered CCK also produces a hyperglycemia with a concomitant elevation in plasma glucagon [53]. The biologically active moiety of CCK is its C-terminal octapeptide which is synthesized and stored in duodenal mucosa and released by the arrival of food in the duodenum. The peptide also stimulates cells of the pancreas and gall bladder when administered systemically [80]. CCK has been identified in the mammalian brain by radioimmunoassay, with its receptors distributed differentially in varying concentration in the cortex, olfactory bulb, hypothalamus, thalamus, caudate nucleus and cerebellum [ 12, 14,25,30]. In addition, anatomical studies have revealed that discrete pathways as well as terminal neurons of the brainstem and cerebrum clearly contain CCK [19,20]. Not only does CCK co-exist with dopamine in neurons of the mesolimbic system [28] but this peptide can affect the metabolism of dopamine and other amines in subcortical structures (e.g., [16,32]). Interestingly, both CCK-8 and CCK-4 differentially alter the binding characteristics of both dopamine and 5-HT receptors in the brain [l]. Investigations into the possible mechanisms by which CCK could act centrally began initially in our laboratory on a pharmacological basis. When CCK was applied bilaterally, but not unilaterally, to sites in the hypothalamus that were reactive to NE in terms of its inducing spontaneous feeding, the ingestive response was markedly attenuated [42]. Curiously, however, CCK enhanced the NE-induced feeding response at certain sites in the anteromedial hypothalamus. When CCK was injected intraperitoneally, the feeding caused by NE injected into the hypothalamus also was significantly suppressed 1421. In physiological experiments, we

588

MYERS EZ’ AL.

SATIATED

FASTED

VENTROMEDIAL

N.

2.0. ii $. 1.5.

PERFUSION

SITE

d cK 1.0. iii I z 0.5.

-40

0

40

80 -40 0 TIME (MINUTES)

40

80

FIG. 3. Proportion [3H]-NE release from perfusion site in AP 5.0 within VMN ofa representative rat when fully satiated (left) or fasted for 22 hr (right). CCK (0.05 Fg/min) was perfused (histological inset) at zero time and excess K+ ions (2.5mM) at 40 min time (right). Asterisk (*) denotes statistically significant change (pSO.01).

found that when CCK was injected systemically, the release of 14C-NE was enhanced at sites in the medial and rostra1 hypothalamus involved in the noradrenergic feeding response [64]. Further, the infusion of a nutrient substance directly into the duodenum produced either an enhancement or reduction in NE release from circumscribed sites in the lateral or medial hypothalamus, respectively [63]. This observation provided direct functional support for a CNS noradrenergic link in the vagal-tierent hypothesis of CCK’s mediation [80]. Related findings have shown that lesions of the dorsomedial and paraventricular nuclei of the rat suppress the satiety effect of CCK [4,7]. During the course of further experiments with CCK perfusions in the unrestrained rat, we discovered an inexplicable inconsistency in the pattern of NE release from the regions of the hypothalamus involved in the nomdrenergic feeding mechanism. After ruling out such factors as time of day, light-cycle, activity of the animal, and external stimuli such as odor [67], we found that the state of hunger or satiety determined the nature of CCK’s action on noradrenergic neurons in the hypothalamus [61]. Not only does the nutritional status of the rat apparently govern the nature of NE activity, as reflected in its presence in perfusate, but the differential response to CCK is anatomically dependent as well. CCK Perfusion In the present experiments, 21 rats were tested; however, data on three animals could not be included because of their extra-hypothalamic perfusion sites. Each locus of perfusion in the hypothalamus of the remaining 18 rats was labeled with [3H]-NE, 20-30 min prior to the first push-pull perfusion, as described under the General Method section. The flow rate was 25 pl/min with each sample of perfusate collected over a 5.0 min interval. Although three doses of CCK were employed, 50 ng, 100 ng and 312 ng/min, our preliminary findings revealed the absence of any consistent dose response relationship. Therefore, a single dose of 50 ng/min

of CCK or 2.0 ng/pl was used in most of the experiments described. Altbot.@ control wash-out curves were obtained during

the fasted (n=24) and the sated (n= 11) states, there were no significant differences between them. The control mean values rS.E. were plotted for the sample before the baseline and for >6 samples following the perfusion with CCK or other test compound. Each push-pull perfusion experiment was carried out between 900-l 100 hr or 1400-1600 hr on each day. Anatomical

Analysis of CCK’s Action

An anatomical analysis of each of the sites of perfusion in 18 rats revealed that 30 of the total number of 62 sites or 48% were reactive to the localized push-pull perfusion of CCK in terms of either an enhancement or suppression of [3H]-NE efflux. The effect of food deprivation on the reactivity of sites in the hypothalamus revealed that CCK was slightly more active in altering the output during the fasted state. Whereas CCK altered the efflux of [3H]-NE at 13 of 29 sites or 45% in the sated rat, the pattern of efflux was altered in 17 of 33 sites or 5% in the food-deprived animal. An anatomical map is presented in Fig. 1 which portrays the distribution of perfusion loci from AP 4.0-6.5 within each of which 2.0 ng/pl CCK was perfused in rats which were either satiated or fasted. At medial hypothalamic sites encompassing the PVN, the region posterior to the PVN, and the VMN of the fully satiated rat, CCK evoked a consistent and significant release of [3H]-NE. As shown in Fig. 1 (right), CCK enhanced the release of [3H]-NE in 9 of 16 or 56% of the sites of perfusion within sagittal planes medial to the columns of the fomix. However, as shown in Fig. 1 (right), t3H]-NE efflux in loci located more laterally exhibited no change in response to the perfusion of CCK. On the other hand, it is clear from this mapping study that in the fasted animal (Fig. 1, left) CCK enhanced the release of [3H]-NE in all but two of the lateral sites within the col-

CCK AND NOREPINEPHRINE

589

RELEASE

FASTED

SATIATED LATERAL

CLK

M

-40

HYPOTHAL.

PERFUSION SITE (AP 6.0)

.T

.

.

80 -40 0 TIME (MINUTES)

.

.

.

e

40

FIG. 4. Proportional [3H]-NE release from perfusion site in AP 6.0 within LH of a representative rat when fully satiated (left) or fasted for 22 hr (right). CCK (0.05 wuglmin)was perfused (histological inset) at zero time. Asterisk (*) denotes statistically significant change (pct!i.Ol).

umns of the fomix or those distributed more laterally in AP 6.0, 5.5, 5.0 and 4.5. Conversely, at 3 of 15 sites, or 20%, located in medial hypothalamic structures, CCK suppressed the output of [3H]-NE during the fasted condition. Although a site in DMN released [3H]-NE during the perfusion of CCK, the remaining 11 sites tested were unaffected by the peptide. Table 1 presents a summary analysis of the anatomical sites of perfusion in individual rats and the alteration in [3H]-NE efflux in response to CCK perfusion during the satiated or fasted condition. The sites are divided into three general morphological regions: (1) lateral hypothalamus including all sites lateral to the columns of the fomix; (2) VMN and PVN; and (3) other sites including the DMN. Table 1 shows that changes in the [3H]-NE eMux pattern depended not only upon the frontal AP plane but also on the depth of perfusion in an individual rat. To illustrate, in 8 or 9 lateral hypothalamic sites perfused under the fasted condition, CCK evoked a release of [3H]-NE. At 4 of 5 sites in both the PVN and VMN, the release of [3H]-NE in response to CCK was enhanced during the satiated condition; however in 3 of the 6 medial sites tested in the fasted rat, the release of the catecholamine was suppressed by CCK. Table 1 also shows that at the junction of the DMN-VMN, LH-PVN, LH-DMN, as well as in the optic tract, CCK typically enhanced [3H]NE release whether the animal was satiated or fasted. It is particularly notable also that at 3 of 4 sites in which the DMN was perfused (Table l), CCK evoked a release of NE during the fasted condition, suggesting a functional similarity of this nucleus to that of the lateral hypothalamus. Patterns

of CCK-Induced

NE Release

in Hypothalamus

Distinct differences in the pattern of efflux of [3H]-NE in response to the perfusion of CCK within the region of the PVN is presented in Fig. 2. During the satiated state, CCK significantly enhanced [3H]-NE release at zero time after which the slope of the efflux paralleled that of the control.

TABLE 2 PERCENT CHANGE IN CCK EVOKED INCREASE (t) OR DECREASE (J) IN NE RELEASE FROM HYPOTHALAMUS OF RATS FASTED (22 HR) OR SATED Anatomical Structure

Sated

Fasted

LH PVN VMN

100% 0 100% t 86% t

8% t 50% J 100% 0

25% t 44%?

100% t 55% t

DMN Other (MM, ARC, OT, AVN, MFB, PAG, PH)

Conversely, CCK perfused at the same site in the same animal which was fasted for 22 hr suppressed NE efflux during the interval of perfusion. Thereafter, the perfusion of excess K+ ions, as depicted in Fig. 2, evoked a strong release of NE within the perfusion site. Within the region of the VMN of a satiated rat, CCK induced a marked output of NE at a perfusion site within coronal plane AP 5.0. As illustrated in Fig. 3, this significant enhancement persisted also during the interval following the perfusion with CCK. However, when the same animal was fasted, CCK was without effect on r3H]-NE output in the VMN. Once again, excess K+ ions did evoke an efflux of the catecholamine from this site of perfusion (Fig. 3). Figure 4 illustrates the opposite effect produced by CCK when a site in the lateral hypothalamus was perfused with the octapeptide. In the satiated rat, CCK delivered in the lateral area in coronal plane AP 6.0 failed to evoke an immediate efflux of [3H]-NE. However, the perfusion of the peptide at the same locus in the fasted rat evoked a significant efflux of the catecholamine. A composite summary is presented in Table 2 of the per-

590

MYERS ET AL. SATED A PROGL (1.2 pg/@l) . CCK (6 NG/p!) 0 PROGL + CCK

1

= CONTROL

AP 6.5

I_ -10

0

10 20 30 TIME (MINUTES)

40

FIG. 5. Proportional [“HI-NE release from perfusion site in AP 6.5 of representative rat when fully satiated. At zero time, substance was perfused (histological inset) as follows: proglumide (1.2 @g/WI) (A); CCK (6.0 ng/Fl) (0); proglumide plus CCK (0) combined in perfusate in concentrations of 1.2 pg and 6.0 ng/pl, respectively.

cent changes anatomical

in the evoked release of NE from different

structures

in the hypothalamus

during CCK per-

fusion. As shown in Table 2 both VMN and PVN exhibited an enhanced efflux during the satiated state, whereas in the lateral hypothalamus, i.e., region of tissue lateral to the columns of the fornix, there were no changes in any of the sites tested. Conversely, in 8% of the sites within the lateral hypothalamus an enhanced efflux occurred when the animals were fasted. During the same condition, 50 percent of the sites in the PVN showed a supression of [3H]-NE release with no effect shown in VMN. Table 2 shows also that in the DMN and adjacent structures, evidence of catecholamine effux was generally noted in all regions in both fasted and sated conditions but in varying percentages. Antagonism

qf’ CCK’s Action krith Proglumide

A well-known drug used clinically in Europe for the treatment of ulcer patients, proglumide, is a competitive antagonist of gastrin in the stomach and duodenum [34]. Proglumide is known also to bind to CCK receptors and thus is believed to be an inhibitor of CCK’s pharmacological action [22]. In addition, proglumide can block selectively the CCK-induced excitation of dopaminergic neurons in the rat’s brainstem [6]. Therefore, a major question has arisen concerning the potential effect of the local blockade of CCK receptors in noradrenergic systems subserving hunger and satiety. The present experiments were carried out with the fully satiated rat. Proglumide was perfused in a concentration of

1.2 &PI alone and in combination with CCK at sites reactive to CCK in terms of [3H]-NE efflux. As shown in Fig. 5, proglumide perfused within the PVN at coronal plane AP 6.5 (histological inset) failed to cause an enhanced efflux of NE, and in fact suppressed the output of the catecholamine. On the other hand, CCK in a concentration of 6.0 ng/pl, perfused at the same site evoked the typical efflux of [3H]-NE (Fig. 2). However, when the same concentrations of both proglumide and CCK were perfused together at the same site in the PVN, the output of [3H]-NE was reduced to a level below that of the control. Thus, the effect of combined perfusion of proglumide plus CCK was virtually identical to that produced by proglumide alone. These results suggest that the CCK antagonist could in itself interfere with the action of CCK on noradrenergic neurons in the PVN. Alternatively, proglumide may exert a direct action on post-synaptic receptors of noradrenergic neurons particularly in view of the suppression of the pre-synaptic [3H]-NE release during perfusion of the CCK antagonist in the PVN (Fig. 5). Since proglumide and another CCK receptor antagonist, benzotript, stimulate feeding when they are microinjected directly into the PVN [ 131, it would appear also that CCK receptors within this nucleus are either involved in the feeding response or that a noradrenergic-CCK receptor link is required for the mediation of satiety in the medial hypothalamus. In another series of experiments currently underway in our laboratory, CCK and proglumide have been perfused at sites in the preoptic area which are also reactive to NE in terms of induced feeding. Samples of perfusate were collected before and during separate perfusions of either CCK, in a concentration of 6.0 ng/pl, or proglumide, in a concentration of 1.2 ~g/~l. Samples of perfusate were assayed by HPLC-EC in this case, according to procedures described under the General Method section. Figure 6 presents an HPLC chromatogram of two samples of perfusate collected from the site depicted in the histologtcal inset. The profile of the amines and their metabolites is characterized in response to CCK perfusion in Fig. 6 (left). Under these conditions, DA together with catecholamine metabolites was the only catecholamine clearly detected: S-HT and its metabolites were also identified in a range which can be compared to the internal standard (IS). HOWever, when proglumide was perfused at the same site (Fig. 6 right), the efflux of DA tended to be lower without a notable alteration in DOPAC levels; nevertheless, an elevation in the efflux of 5-HT was produced by proglumide. Simultaneously, the 5-HIAA and 5-HTOL peaks were lower after perfusion with proglumide in contrast to that during CCK perfusion, relative to the internal standard (Fig. 6 right). Figure 7 presents the results of the CSF sample (left) as well as the profile of standards assayed by HPLC-EC. The

internal standard in each case was isoproteronol which eluted at 21 min. As shown in Fig. 7 (left), the dopamine metabolite, DOPAC, elutes in the highest concentration relative to the internal standard. Overall, these results with HPLC-EC analysis strongly suggest that proglumide not only can act as a CCK receptor antagonist but also can exert a profound functional influence on the activity of 5-HT containing neurons as well. Although these results are preliminary in nature, it is likely that future investigations will necessarily have to take into consideration the serotonergic system in terms of the local neuronal effects exerted antagonists.

by

both

CCK

and

its

pharmacological

CCK AND NOREPINEPHRINE

591

RELEASE

CSF+CCK (6ngluL)

PERFUSION SITE

c

(AP 7.5)

I:

5

l-

3

10

I

*

15

20

25

30

I

5

10

15

20

25

30

35

TIME (MINUTES)

FIG. 6. Representative HPLC-EC chromatograms of perfusion samples collected from site in AP 7.5 of a representative, satiated rat. Left: CCK (6.0 ng/pl) was added to the CSF perfusate; Right: proglumide (1.2 ~li//.J) was added to the perfusate. Isoproteronol as internal standard (IS) was 8.0 picograms with full scale deflection of 0.2 nanoamps. Abbreviations are: DA: Dopamine; DOPAC: Dihydroxyphenyl acetic acid; EPI: Epinephrine; HVA: Homovanillic acid; NE: Norepinephrine; NMN: Normetanephrine; S-HIAA: S-Hydroxyindole acetic acid; 5-HT: 5-Hydroxytryptamine; 5-HTOL: 5-Hydroxytryptophol; 5-HTP: 5-Hydroxytryptophan; VMA: Vanillylmandelic acid.

CSF

STANDARDS

r-i It

ri :

5

IO

15

20

25

30

35‘

FIG. 7. HPLC-EC chromatogram of a CSF perfusate (left) collected from same site of the satiated rat as in Fig. 6. Right: chromatogram of standards with 8.0 picograms isoproteronol (IS) as internal standard and full scale deflection of 0.2 nanoamps. Abbreviations as in Fig. 6.

592

MYERS ET AL. TABLE 3

NT AFFECTS NE RELEASE -A,V FASTED l

PERCENT OF NEUROTENSIN EVOKED INCREASE (t) OR DECREASE (1) IN NE RELEASE FROM DIENCEPHALIC PERFUSION SITES OF RATS EITHER FASTED (22 HR) OR SATED (N=5-81-,

, --SATED

Anatomical Structure

Sated

Fasted

LH PVN VMN

J (50%) t (1m) T (8w)

T (75%) J (29%) J (29%)

Fornix POA ARC DMN

t ? r 0

1 (33%‘) J (SO?+) t (50%) t (20%)

pre-treatment with monoamine chelating agent, EGTA, given prevent the effect of the peptide reverses the poikilothermia agent [66].

P 5.0 FIG. 8. Anatomical “mapping” of hypothalamic sites at which NT (0.05 or 0.01 pg/pl) was perfused in rats which were either 18-22 hr food-deprived (left) or fully satiated (right). Release of [“HI-NE from each site denoted as increased (A), decreased (v) or unchanged (0).

NEUROTENSIN

“MODULATES”

NE ACTIVITY

Neurotensin (NT) has been considered to be a putative satiety hormone following the original report that the systemic infusion of the peptide in the rat suppressed the animal’s intake of food [ 181. This corresponded to the finding that eating alters circulating neurotensin-like immunoreactivity in the human subject 1491. Not only is NT present endogenously in both the gastro-intestinal tract and brain, but it also has been identified in relatively high concentrations in the PVN and VMN of the hypothalamus [29,88]. In addition, this tri-decapeptide can cause an efflux of NE from slices of hypothalamic tissue [70]. When NT is infused directly into the hypothalamus of the food-deprived rat, the normal eating response is substantially reduced with little or no effect on the concomitant intake of water or other responses [24,26,81]. Recent experiments in our laboratory suggest that NT also can disrupt other aspects of energy metabolism. In fact, the poikilothermic action of NT given by the ICV route, in which the core temperature of the rat simply follows that of the ambient temperature, indicates that NT may exert a broad spectrum of functionally incapacitating effects [62]. Although this thermolytic action of NT is not blocked by ICV

(60%) (75%) (50%) (100%)

antagonists [36], the calcium also 1CV prior to NT, does [62] in much the same way as it produced by an anesthetic

An intriguing question surrounding the local mechanism of action of neurotensin (NT) at the level of the hypothalamus is raised by its pharmacological suppression of food intake when applied to the PVN and VMN of the fasted rat [24,81]. Particularly fascinating is the issue surrounding the relationship between NT and those hypothalamic nuclei containing noradrenergic neurons, which are affected so markedly by CCK. In the present experiments the same experimental principles and bench-top procedures were used to examine the local action of NT as those used for CCK. Push-pull perfusion guide cannulae [56] were implanted stereotaxically in a total of 13 rats at loci which were generally homologous to those tested in the CCK experiments. Again the sites of perfusion, which were located in coronal planes from AP 5.0 to 6.5. were labeled with [RH]-NE. Following a counterbalanced design, the sites were subsequently perfused with an artificial CSF when the animals were either satiated with food or fasted for a period of 18-22 hr.

Overall, the perfusion of NT within both lateral and medial hypothalamic regions of the rat produced effects which were very similar to those observed with CCK. An anatomical analysis is presented in Fig. 8 of tissue sites that were reactive to the presence of NT in terms of either an enhanced or suppressed release of [zH]-NE. In the fasted animals, those sites lateral to the descending columns of the fornix exhibited an enhanced release of NE during their perfusion with NT in a concentration of 0.05 or 0.01 pgpl [61]. However, at certain sites of perfusion medial to the fomix, the release of ISH]-NE was suppressed by this peptide. In the same animals which were tested in the satiated condition, an opposite result once again arose. That is, within medial hypothalamic sites of perfusion extending from AP 5.0 through AP 6.5, NT enhanced the efBux of 13H]-NE. However, within the classical lateral hypothalamic “feeding” area 1841of coronal plane AP 5.5, the perfusion of NT in the sated rats inhibited the efflux of 13H]-NE.

CCK AND NOREPINEPHRINE

593

RELEASE

FASTED AP 6.5

AP 6.5

0 -15

. 0

I

I

1

15

30

45

TIME (MINUTES) FIG. 9. Proportional

pH]-NE

release from perfusion sites in

AP 6.5

of two representative rats when 22-hr foodideprived. At zero time, 10.0 ~g/~I 2-M? were added to sample (A) perfused in medial hypothalamus (top) or in LH (bottom). (Modified from McCaleb ef al. 1441.)

Table 3 presents a composite analysis of the percent increase or decrease in [3H]-NE release from hypothalamic sites of NT perfusion in rats which were either fasted or fully satiated. In general, the results were similar to those of CCK with the exception that only in the DMN was there an absence of an effect of the peptide on NE activity. Interestingly, in both PVN and VMN of the fully satiated rat, the effects of CCK and NT were identical. Although NT suppressed NE activity in 50 percent of sites within the lateral hypothalamic region of the satiated rats, CCK failed to affect the efflux of the catecholamine (Table 2). In general, NT caused an enhanced release of NE within the fomical area, pre-optic area and the arcuate nucleus in the satiated animal; however, in the fomix and pre-optic area of the fasted rats, NT tended to suppress the eMux of the amine (Table 3). Taken together, these results support the view that NT can serve to modify the local activity of noradrenergic neurons in the feeding system. Because of the similarity of effect of CCK and NT, it is likely that the tri-decapeptide could also be a modulator of catecholamine-containing neurons which comprise the hunger-satiety system [40]. GLUCOSE-INSULIN:

INTERACTION WITH NORADRENERGIC SYSTEMS

In terms of the profile of factors which affect the feeding mechanism [581, two well known substances believed to act

within the central nervous system, 2-deoxy-D-glucose (2-DG) and insulin both induce spontaneous feeding (see review in [44]). In recent experiments, we found that the perfusion of either 2-DG or insulin within a large number of hypothalamic sites either augments or reduces the release of NE. Because 2-DG acts peripherally to evoke spontaneous feeding in a wide variety of species [69] by virtue of its glucoprivic action, this carbohydrate molecule would be expected to exert differential effects on the lateral and medial hypothalamic neurons. In one series of experiments [44], 2-DG was perfused in the hypothalamus of rats which were food-deprived for 18 hr. Figure 9 illustrates the results of the localized effect of a perfusion of 10 &$ of 2-DG at medial and lateral hypothalamic sites within coronal plane AP 6.5. In Fig. 9 top, it can be seen that 2-DG evokes the release of NE during the interval of its perfusion at a perifomical site. However, NE efflux was reduced initially and then enhanced by an identical perfusion of 2-M; at a site within the lateral hypothalamic area just dorsal to the optic tract. In a series of new experiments currently underway in our laboratory, the effect of 2-DG as well as insulin is being tested in the satiated rat. Once again, the same physiological parameters are employed except that in the latter case the animal is provided with food freely available throughout the experiment. The perfusion of 10 &PI 2-DG within the PVN of the satiated rat enhances significantly the eIIIux of [3H]NE, but within the lateral hypothalmic area induces mixed effects on catecholamine activity. On the other hand, insulin perfused in a concentration of 4.0 mU/pl enhanced the effux of [3H]-NE within the lateral hypothalamus of the fasted rat but suppressed its release within the PVN of the sated rat. The circumscribed effect of 2-DG within the PVN of the satiated animal suggests that a substance which induces feeding by virtue of a glucose deficit produced regionally may act also via the noradrenergic neurons underlying feeding and satiety [43,44]. Insulin affects the PVN pharmacologically in a manner opposite to that of the two putative satiety hormones, CCK and NT. Metabolically, it would appear that insulin should, in fact, serve to satiate the animal when acting locally in the region of the lateral hypothalamus but produce an inhibition of satiety when acting at the levels of the PVN or VMN. GENERAL DISCUSSION

The most important finding of this series of experiments is the state-dependent nature of the effect of a peptide or other substance on the in vivo activity of a catecholamine neurotransmitter. Of special significance is the fact that in the conscious and freely-moving animal, the measure of NE in perfusate collected from the hypothalamus can reflect the unique augmentation or attenuation of the amine’s release by CCK or NT. Either response is contingent solely upon the nutritional status of the rat, fasted or satiated. Equally remarkable is the distinct anatomical separation of the patterns of transmitter release produced by the peptide within the hypothalamus itself. The functional similarity of the PVN and VMN suggests that a coordinated and integrated physiological mechanism exists in the NE-containing nerve cells within these two nuclei. In relation to their individual activity involving neuronal NE, either a condition of fasting or nutritional balance is recognized by the PVN and VMN in an identical manner. The condition of deprivation, plus the peptide either suppresses NE activity or exerts little

594

if any quantifiable effect on the catecholamine’s efflux. Although Leibowitz and colleagues report differences between the PVN and the VMN in specific NE binding characteristics [31], the precise mechanism which differentiates them is not clear at this point. Not unexpected is the clearcut anatomical differentiation of NE reactivity between the two medial structures and that of the classical lateral hypothalamic area which is generally agreed to subserve the activation of feeding 126, 75, 86, 891. An inverse response in the pattern of NE efflux to that evoked in the medial hypothalamus can occur as a result of the local delivery of CCK, NT, glucose and insulin to portions of the lateral hypothalamus extending from its caudal to rostra1 aspect. Presently it is unknown whether a similar kind of change in NE release would characterize other structures implicated in peptide-induced feeding or satiety [82,90]. On the basis of this anatomical distinction, it is postulated that neuroactive peptides serve as neuromodulators of catecholamine transmitters in both of the pivotal regions in the hypothalamus underlying ingestive behavior [36,84]. This assumption is based on several pieces of evidence as follows. Electrophysiologically, it is apparently that NE is predominately an inhibitory neurotransmitter of cells in the brain [2,39]. Consequently, an infusion of NE into either of the two medial hypothalamic nuclei would inhibit those neurons which underly the satiety mechanism and the satiated animal would eat [27]. In support of this viewpoint are earlier physiological results which show that NE efflux increases within the medial hypothalamic region of the fasted rat as it consumes food [48]. In the fasted animal, the local action of CCK or NT within these cells, therefore, would reduce presynaptic release of NE. In turn, the status of satiety is subsequently not inhibited and the animal would not feed. Indeed, the presence of the peptides in either PVN or VMN, where they serve to inhibit feeding [42,90], does attenuate the local efllux of NE. In contrast, the presence of CCK or NT in the lateral hypothalamus of the fasted animal would be expected to enhance the release of NE from noradrenergic neurons in order to inhibit feeding. This is precisely what happens. Although the results obtained with both medial and lateral hypothalamic perfusion of either peptide in the satiated rat is not entirely clear in regard to the enhanced or suppressed release of NE, it is conceivable that certain of the peptides should augment the intake of food by activating the release of NE from the medial “satiety neurons” [40,71]. In addition to the eating response elicited by neuropeptide Y applied to the PVN and VMN [82], CCK infused within medial hypothalamic sites tends to augment the feeding response produced by the local microinjection of-NE (421. Whether these peptides could play a dual role in terms of activating the intake of food is a question that remains to be explored [79]. Nevertheless, CCK, NT or another peptide could operate in the hypothalamus by one of several mechanisms: (1) enhance the presynaptic release of NE into the synaptic cleft; (2) produce a shift in the depolarization properties of the catecholaminergic neurons by virtue of local membrane effects; (3) retard selectively the presynaptic or postsynaptic uptake of the catecholamine; (4) modify the intracellular metabolism of the amine in noradrenergic neurons; and (5) exert a direct effect on pre- or postsynaptic receptors within catecholaminergic nerve terminals. Insofar as the glucose-insulin mechanism is concerned in the rat, the release of insulin associated with a meal involves a vagal-visceral neuronal pathway; it is controlled centrally,

MYERS ET AL. at least in part, by the VMN [85] without which the release of insulin is blunted. Precisely what part the lateral hypothalamus plays in the insulin response is currently unresolved. However, a lesion of the lateral hypothalamus attenuates insulin-elicited feeding; further, insulin facilitates the uptake of 2-DG in the perifornical area of the rat’s hypothalamus but not in the VMN [33]. Of considerable significance is the recent observation that insulin binding to receptors in the lateral hypothalamus of the rat is generally much lower than that seen in the medial region [50]. After prolonged restriction of food, receptor binding in the lateral region of the rat remains unchanged but is substantially reduced within the medial hypothalamus. Consequently, insulin could function by way of different populations of hypothalamic receptors in these two areas of the hypothalamus, providing one form of metabolic feedback signal to the central controller for hunger [91]. After NE is infused directly into the lateral hypothalamus, plasma insulin is elevated in the rat, but when the catecholamine is infused into the VMN, circulating levels of both insulin and glucose increase [83]. These findings imply that neurons of the medial and lateral hypothalamic regions are differentiated at the receptor level on the basis of their carbohydrate sensitivity. In this connection, the feeding caused by a 2-DG-induced deficit in circulating glucose [69] ostensibly requires an afferent vagal projection to the medial hypothalamus 1761. Thus, the increase in the efflux of NE in the PVN or VMN produced by 2-DG given peripherally could be due to the stimulation of vagal afferent pathways (431. Accordingly, the local release of NE during the hypothalamic perfusion of 2-DG could be triggered by a regional depletion in glycogen stores due to a circumscribed reduction in the level of glucose 1431. Consequently, it is envisaged that a change in NE release, which is so clearly differentiated anatomically in the hypothalamus. reflects the direct action of an excess or deficit in nutrient level on the respective noradrenergic cells in the lateral and medial regions of the hypothalamus. One clear-cut conclusion can be deduced from the present experiments. It is now essential that the local endogenous activity of CCK, NT and other centrally acting peptides, such as neuropeptide Y, should in themselves be investigated in the conscious animal. Presumably, the nutritional status of the animal can alter the synthesis, accumulation. sequestration, and release of a neuropeptide in the hypothalamic parenchyma. Hence, the kinetics of release of NE and other neurotransmitters such as 5-HT or the amino acids could co-vary with a change in peptidergic activity, particularly as a specific peptide modulates a key transmitter. In this context a simple pharmacological observation on peptide-induced feeding or its termination is really an “empty observation” without a concomitant verification of its evoked release at the same site by a stimulus of physiological relevance. What specific substance or profile of factors comprises the stimulus complex which impinges on a given peptide is unknown. Glucose, insulin, glucagon, free fatty acids and amino acids could certainly be viewed as likely candidates (1.5, 68, 77, 921 which functionally influence the kinetics of the neuropeptide. Therefore, the cascade of physiological events requiring humoral factors, such as the endogenous peptides in the hypothalamus will have to be elucidated during different nutritional states. Such pivotal knowledge on the interaction of these substances in the brain with factors circulating peripherally will help explain the mechanisms underlying the elaboration of the feeding response itself [581.

CCK AND NOREPINEPHRINE

595

RELEASE

At this point, NE still predominates over the center of the neurochemical “stage” of hypothetic function. Our experiments point to the difficult physiological question pertaining to endogenous changes in CCK, for example, at the hypothalamic level. In addition to its effect on presynaptic release, does CCK also alter receptor protein or modify the synthesis, turnover and storage of the neurotransmitter? Clearly, the new era of neurochemical complexity subdues the early and attractive concepts of neurochemical “coding” [57j delineated for the onset of feeding behavior and its termination. Presently, it would be difficult to accept as quintessential a relatively simplistic notion that NE serves as the only substance of such a “code” in the hypothalamus. To understand how a peptide or other neurochemical factor acts in a unique way as a neuronal determinant of the states

of hunger and satiety now constitutes scientific challenge.

a new and profound

ACKNOWLEDGEMENTS This research was supported in part by National Science Foundation Grants BNS-78-24491 and BNS-84-10663 to R.D.M., by U.S. Spanish Joint Committee (J.M.P.) and Junta de Andalucia (J.M.R.F.). The authors are particularly grateful to Dr. Amir H. Rezvani for his cogent suggestions; Drs. Jacqueline Crawley and D. Lucania for kindly providing proglumide and CCK, respectively; W. Holahan, Leslie Gurley-Orkin, Rebecca Nunn and Carol Ensor for histological, graphic and other technical assistance; Kevin T. McManus for HPLC-EC analysis of samples of per&ate; and Victoria Staten for the preparation of the manuscript.

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15. Frohman, L. A. and L. L. Bemardis. Effect of hypothalamic stimulation on plasma glucose, insulin, and glucagon levels. Am J Physiof 221: 1596-1603, 1971.

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34. Knoll, B. and J. Knoll. The selectivity of the anorectic effect of satietin. PO/ I Pharmacol Pharm 34: 17-23, 1982. 35. Lee, T. F. and R. D. Myers. Calmodulin-induced feeding in the satiated cat: Evidence for the involvement of calcium and norepinephrine in the brain. Brain Res Bull 12: 71-76, 1984. 36. Lee, T. F., J. R. Hepler and R. D. Myers. Evaluation of neurotensin’s thermolytic action by ICV infusion with receptor antagonists and a Ca++ chelator. Pharmacol Biochem Behal, 19: 477-481,

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37. Lee, T. F., D. M. Denbow, S. E. King and R. D. Myers. Unique . ^ . . _. contnbutlon of catecholamme receptors m the brain of the cat underlying feeding. Physiol Behav 29: 527-532, 1982. 38. Lee, T. F., F. Mora and R. D. Myers. Dopamine and thermoregulation: An evaluation with special reference to dopaminergic pathways. Neurosci Biohehal* Rev 9: 589-598, 1985. 39. Lee, H. K., C. T. Chai, P. M. Chung and C. C. Chen. Response of temperature-sensitive medullary neurons to acetylcholine, norepinephrine and 5-hydroxytryptamine. Brain Res Bull 2: 381-388, 1977. 40. Leibowitz, S. F. Neurochemical systems of the hypothalamus: Control of feeding and drinking behavior and water-electrolyte excretion. In: Hundbook of the Hypothalumus. vol 3, edited by P. .I. Morgane and J. Panksepp. New York: Marcel Dekker. 1980. pp. 299-437. 41. McCaleb, M. L. and R. D. Myers. Striatal dopamine release is altered by glucose and insulin during push-pull perfusion of the rat’s caudate nucleus. Bruin Res Bull 4: 651-656, 1979. 42. McCaleb, M. L. and R. D. Myers. Cholecystokinin acts on the system” involved in feeding. hypothalamic “noradrenergic Peptides 1: 47-49, 1980. 43. McCaleb, M. L. and R. D. Myers. 2-deoxy-D-glucose and insulin modify the release of norepinephrine from rat hy’pothalamus, Am J Physiol 242: R596R601, 1982. 44. McCaleb, M. L., R. D. Myers, G. Singer and G. Willis. Hypethaiamic norepinephrine in the rat during feeding and perfusion with glucose, 2-DG or insulin. Am J Physiol236: 312-321, 1979. 45. Mann, J. F. E., R. Boucher and P. W. Schiller. Rotational syndrome after central injection of C-terminal 7-peptide of cholecystokinin. Pharmacol Biochem Behav 13: 125-127, 1980. 46. Martin. G. E. and R. D. Mvers, unpublished observations, 1971. 47. Martin, G. E. and R. D. My&s. -Action of intrahypothalamic 6-hydroxydopamine on motivated responding for food and water in the rat. Pharmacol Biochem Behal’ 2: 393-399, 1974. 48. Martin, G. E. and R. D. Myers. Evoked release of (“‘C) norepinephrine from the rat hypothalamus during feeding. Am J Physiol 229: 1547-1555, 1975. 49. Mashford, M. L., G. Nilsson, A. Rakaeus and S. Resell. The effect of food ingestion on circulating neurotensin-like immunoreactivitv (NTLI) in the human. Actor Phvsiol Stand 104: 244-246, 1978: 50. Melnyk, R. B. and J. M. Martin. Starvation-induced changes in insulin binding to hypothalamic receptors in the rat. Actrr Endocrinol

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